Actinobacteria isolation from marine environments

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Pre-treatment methods of marine samples

Pre-treatment methods for the isolation of marine Actinobacteria have been applied to eliminate the growth of terrestrial actinobacteria, gram-negative bacteria and fungi (1). These methods include drying the samples using laminar air flow hood, mixing the samples with seawater, applying UV irradiation, applying high frequency radiation and the use of certain antibiotics to kill gram negative bacteria.

Drying marine samples with laminar air flow hood

Several drying pre-treatment methods can be used to enhance the isolation of Actinobacteria from marine environments (2):

  1. Drying sediment overnight in a laminar flow hood until clumping occurs. Press a sterile foam plug against the sediment sample and onto the surface of an agar plate making a serial dilution (2).

(2#) Drying sediment overnight in a laminar flow hood until clumping occurs. Dilute dried sediment with 5 ml of sterile seawater and mix well. Add 50 µl of the solution onto an agar plate and spread with an spreader (2).

Mixing the samples with seawater

Media can be enriched with 2% of seawater or samples can be added directly grown in sterile (autoclaved) seawater (3).

Applying UV irradiation

Directly apply UV‐irradiation to the sediment suspension (5 ml) for 30 s. Use a wavelength of 254 nm and leave a distance of 20 cm (4).

Applying high frequency radiation

Apply superhigh frequency radiation directly to sediment samples by placing the sediment solution in a Eppendorf tube and microwave it for 45 seconds at 2460 MHz and 80 W (4).

Dilute / Heat

Add dry sediment to 3 mL of seawater (dilutions 1:3 or 1:6), heat it to 55°C for 6 min, and inoculate 50-70 µL of the resulting suspension onto an agar plate (2).

Media selection

Soil agar (4):

Filtered soil extract (mix 200 g of soil in 1 L of water and boil for 30 min) – 1 L

Agar – 20 g


Soil agar with 3% sea salt added


Modified organic agar 2 Gause (4):

Peptone – 5.0 g

Trypton – 3.0 g

Glucose – 10 g

NaCl – 5.0 g

Tap water – 1 L


Modified organic agar 2 Gause with 3% sea salt added (4)


Mineral agar 1 Gause (4):

Starch‐soluble – 20.0 g

K2HPO4– 0.5 g

MgSO4– 0.5 g

KNO3– 1.0 g

NaCl – 0.5 g

FeSO4−0.01 g

Agar – 20.0 g

Tap water – 1 L


Soy‐bean meal agar (4):

Soy‐bean meal – 3.0 g

KNO3– 0.2 g

K2HPO4– 0.5 g

MgSO4– 0.4 g

Agar 20.0 g

Tap water – 1 L


Soy‐bean meal agar with 3% sea salt added (4)


Yeast‐corn‐starch agar (4):

Starch‐soluble – 10.0 g

Yeast extract – 10 g

Corn meal extract – 10.0 g

NaCl – 2.0 g

Agar – 20.0 g

Tap water – 1 L


Pea‐meal agar (4):

Pea meal – 10.0 g

Glucose – 10.0 g

NaCl – 5.0 g

CaCO3– 1.0 g

Agar – 20 g

Tap water – 1.0 L

Adjust to pH to 7.0 - 7.5 and supplement with nalidixic acid (10 μg ml−1) and nystatin (50 μg ml−1) to inhibit the growth of Gram-negative bacteria and fungi.


M3 (5): Agar – 18 g Monopotassium phosphate – 0.466 g

Disodium phosphate – 0.732 g

Potassium Nitrate – 0.10 g

Magnesium Sulfate heptahydrate - 0.10 g

Calcium Carbonate – 0.02 g

Iron sulfate heptahydrate – 200 µg

Zinc sulfate heptahydrate – 180 µg

Sodium propionate – 0.20g

Nystatin – 50 µg/ml

Nalidixic acid –10 µg/ml

References

1- Subramani, R. and Sipkema, D. (2019) ‘Marine rare actinomycetes: A promising source of structurally diverse and unique novel natural products’, Marine Drugs. doi: 10.3390/md17050249.

2- Jensen, P. R. et al. (2005) ‘Culturable marine actinomycete diversity from tropical Pacific Ocean sediments’, Environmental Microbiology. doi: 10.1111/j.1462-2920.2005.00785.x.

3- Mincer, T. J., Fenical, W. and Jensen, P. R. (2005) ‘Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora’, Applied and Environmental Microbiology. doi: 10.1128/AEM.71.11.7019-7028.2005.

4- Bredholt, H. et al. (2008) ‘Actinomycetes from sediments in the Trondheim fjord, Norway: Diversity and biological activity’, Marine Drugs. doi: 10.3390/md6010012.

5- Maldonado et al. (2005) 'Diversity of culturable Actinobacteria in geographically widespread marine sediments', Antonie Van Leeuwenhoek. doi: 10.1007/s10482-004-6525-0